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Infectious bursal disease virus Vero cell-adapted strain and application thereof

A chicken infectivity, bursa of fabric technology, applied in the direction of antiviral agents, viruses/phages, viral antigen components, etc. High titer, protection of vaccinated animals, long-lasting effect

Inactive Publication Date: 2014-07-16
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the virus titer of IBDV vaccine produced in China is low, and the immune protection efficiency is low; in the production method, the production method of inoculating IBDV in a spinner bottle is still used, and the culture volume is about 0.5L, which cannot be prepared on a large scale

Method used

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  • Infectious bursal disease virus Vero cell-adapted strain and application thereof
  • Infectious bursal disease virus Vero cell-adapted strain and application thereof
  • Infectious bursal disease virus Vero cell-adapted strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 The acquisition and characteristic determination of IBDV virus

[0027] Suspected chicken infectious bursal disease material isolated from chicken flocks in Jiangsu, my country, was used to inoculate specific pathogen-free (Specific Pathogen Free, SPF) virus isolated from chicken embryos. Plaque purification was performed on chicken embryo fibroblasts, and the virus NJ strain was finally obtained through purification and screening of different plaques.

[0028] The NJ strain of the virus was identified for the following characteristics:

[0029] (1) Virus NJ strain amplification: Dilute the virus NJ strain with sterile PBS buffer for 10 3 After doubling, the 10-11-day-old SPF chicken embryos were inoculated by the chorioallantoic membrane route, the inoculation volume was 0.2 mL / embryo, cultured at 37°C, and the allantoic fluid of the chicken embryos was harvested within 36-48 hours.

[0030] (2) EID of the virus 50 : The chick embryo allantoic fluid obtaine...

Embodiment 2

[0034] Example 2 Screening of chicken infectious bursal virus Vero cell-adapted strain

[0035] In this implementation, chicken infectious bursal virus NJ strain was propagated on Vero cells cultured without serum, and a strain of IBDV virus NJ-23 with high virus titer and stable genetic characteristics was obtained through screening by limiting dilution method.

[0036] specific method:

[0037] (1) Vero cells were cultured with IVT medium (serum-free medium, purchased from Gibico) to a confluence of 95%, digested with 0.1% trypsin and centrifuged at 1000rpm, and resuspended in fresh IVT medium . Count the cells according to 8×10 5 96-well plate was laid in cell / well.

[0038] (2) Screening: Dilute NJ strain virus liquid with IVT medium added with whey protein hydrolyzate (Sigma) at a final concentration of 0.25-2.5% (mass percentage concentration), and use 5 dilutions (10 -4 ~10 -8 ) virus liquid infection step (1) spread the monolayer of Vero cells in a 96-well plate, ...

Embodiment 3

[0049] Example 3 Cultivate IBDV virus NJ-23 strain virus liquid on the Vero cells of serum-free microcarrier suspension culture

[0050] The method for cultivating the IBDV virus NJ-23 strain virus liquid on the Vero cells of serum-free microcarrier suspension culture is as follows:

[0051] (1) Serum-free culture of Vero cells: Culture Vero cells in IVT medium. When the cells reach 95% confluence, digest with 0.1% trypsin and centrifuge at 1000rpm, resuspend the cells with fresh IVT medium, and dissolve the cells Transfer to new culture flasks according to a certain ratio, continue to culture until the cells reach 95% confluence, and obtain Vero cells.

[0052] (2) Initial Vero cells were cultured in serum-free microcarrier suspension: the virus maintenance solution was obtained after adding whey protein hydrolyzate at a final concentration of 0.25% (mass percentage concentration) to the IVT medium, and its pH value was 7.1. The Vero cells obtained in step (1) were digested ...

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Abstract

The invention provides an infectious bursal disease virus Vero cell-adapted strain and belongs to the field of bioengineering. The related infectious bursal disease virus Vero cell-adapted strain is named Ck / Jiangsu / NJ-23 / 2008 and has a collection No. of CGMCCNO.8852. The infectious bursal disease virus Vero cell-adapted strain which can efficiently multiply on serum-free cultured Vero cell is finally obtained through wild strain separation, chick embryo passage, Vero cell passage adaption; the infectious bursal disease virus Vero cell-adapted strain is subjected to continuous passage culture on the serum-free cultured Vero cell and TCID50 can be kept to be higher than 108.5 / mL. Virus culture solution is inactivated and prepared into oil emulsion; after the prepared oil emulsion is used to immunize chicken, detection proves that the prepared oil emulsion has good immunogenicity. The infectious bursal disease virus (IBDV) strain and the production process thereof are simple, safe and efficient and suitable for industrial culture.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a Vero cell-adapted strain of chicken infectious bursal virus and an application thereof. Background technique [0002] Chicken infectious bursal disease (Infectious Bursal Disease, IBD) is a highly contagious disease of chickens caused by infectious bursal virus (IBDV). In young chickens aged 3 to 6 weeks, the virus multiplies rapidly in the lymphocytes of the bursa, damages the B lymphocytes of the bursa, and causes severe immunosuppression. After 1980, the disease was introduced into my country and broke out in a large area and continued to be popular, which seriously affected the development of my country's chicken industry. Currently, vaccination is generally used for prevention of the disease. [0003] At present, domestically produced IBDV vaccines usually use chicken embryo fibroblasts to culture and proliferate the virus. Chicken embryo fibroblasts need to add a certain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/12A61P31/14C12R1/93
Inventor 吴培培冯磊禇轩唐应华侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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